Novel method for preparing long-acting drug conjugate through preparation of intermediate

ABSTRACT

Provided is a novel method for preparing a long-acting drug conjugate and a long-acting drug conjugate prepared using the method.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is Continuation of U.S. application Ser. No. 17/627,890filed Jan. 18, 2022, which is a National Stage of InternationalApplication No. PCT/KR2019/008912 filed Jul. 18, 2019.

SEQUENCE LISTING

The content of the electronically submitted sequence listing, file name:Q286375_Sequence_Listing_As_Filed.XML; size: 36,640 bytes; and date ofcreation: Apr. 20, 2023, filed herewith, is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention relates to a novel intermediate for preparing along-acting drug conjugate, a composition including the same, and amethod for preparing the long-acting drug conjugate using the same.

BACKGROUND ART

Physiologically active polypeptides are easily denatured due to lowstability, degraded by proteases in the blood, and easily removed by thekidneys or liver. Thus, in order to maintain blood concentration andtiter of a protein drug containing a physiologically active polypeptideas a pharmacological component, the protein drug needs to be frequentlyadministered to a patient. However, since most protein drugs areadministered to patients in the form of an injection, frequentadministration via injection to maintain blood concentration of thephysiologically active polypeptide causes severe pain to the patientsand increases costs for treatment. To solve these problems, efforts havebeen made to maximize the efficacy of protein drugs by increasing bloodstability of the protein drugs and maintaining the blood concentrationthereof at a high level for a long period of time. However, theselong-acting formulations of protein drugs should not induce immuneresponses in patients while increasing the stability of the proteindrugs and maintaining the titer of the drug at a sufficiently highlevel.

As a method of stabilizing proteins, inhibiting contact with proteases,and suppressing renal clearance, a method of chemically adding a highlysoluble polymer such as polyethylene glycol (hereinafter referred to as“PEG”) to the surfaces of protein drugs has conventionally been used.However, while the method of using PEG may extend in vivo duration ofthe peptide drug by increasing a molecular weight of PEG, the titer ofthe peptide drug significantly decreases as the molecular weightincreases, and a yield may decrease due to low reactivity with thepeptide.

Therefore, as a method for increasing serum half-life, a conjugate of animmunoglobulin fragment and a physiologically active polypeptide hasbeen used, and various studies have been conducted to improvepreparation methods therefor (Korean Patent Application Laid-openPublication No. 10-2014-0109342).

In particular, there has been a steadily increasing need for thedevelopment of efficient processes for preparing a long-acting drugconjugate by simplifying the existing preparation process.

DISCLOSURE Technical Problem

An object of the present invention is to provide a novel intermediatefor preparing a long-acting drug conjugate.

Another object of the present invention is to provide a composition forpreparing a long-acting drug conjugate including the intermediate.

Another object of the present invention is to provide a method ofpreparing a long-acting drug conjugate using the intermediate.

Another object of the present invention is to provide a long-acting drugconjugate prepared by the preparation method.

Technical Solution

One aspect of the present invention provides a novel intermediate.

In an embodiment, the present invention provides a compound having astructure of Formula 1 below or a stereoisomer, a solvate, or apharmaceutically acceptable salt thereof:

X-L1-(OCH₂CH₂)_(n)O-L2-R  [Formula 1]

wherein in Formula 1 above,

X is an immunoglobulin Fc region;

L1 is a straight or branched-chain C₁-C₆ alkylene;

L2 is -a1-CONH—, -a1-NHCO—, -a1-NHCO-a2-, —COO—, -b1-COO—, —COO-b2-, or-b1-COO-b2-, and a1, a2, b1, and b2 are each independently a straight orbranched-chain C₁-C₆ alkylene;

n is from 10 to 2400; and

R is any one selected from the group consisting of2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C₆-C₂₀aryl disulfide, C₅-C₂₀ heteroaryl disulfide, vinyl sulfone, thiol,halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester,and derivatives thereof.

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to the previous embodiment, wherein inFormula 1, L1 is a straight or branched-chain C₁-C₆ alkylene; L2 is-a1-NHCO— or -a1-NHCO-a2-; a1 and a2 are each independently a straightor branched-chain C₁-C₆ alkylene; n is from 200 to 250; and R ismaleimide.

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the X is an immunoglobulin Fc region including a hinge sequence at theN-terminus.

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the X includes a hinge sequence modified to include only one cysteineresidue by deleting a part of an amino acid sequence of amino acidsbelow:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the hinge sequence includes an amino acid sequence of SEQ ID NO: 8(Ser-Cys-Pro) or an amino acid sequence of SEQ ID NO: 9(Pro-Ser-Cys-Pro).

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the L1 is linked to an amine or thiol reactive group located at one endof X.

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the compound has a structure of Formula 2 below:

wherein in Formula 2, n is from 200 to 250.

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the X is an immunoglobulin Fc region derived from IgG, IgA, IgD, IgE, orIgM.

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the X is an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, orIgG4.

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the X is an immunoglobulin Fc region in a dimeric form.

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the X is an immunoglobulin Fc region including an amino acid sequence ofSEQ ID NO: 10.

In the compound or a stereoisomer, a solvate, or a pharmaceuticallyacceptable salt thereof according to any of the previous embodiments,the compound has a size of 40 kDa to 250 kDa.

Another aspect of the present invention provides a composition forpreparing a long-acting drug conjugate including the compound, or astereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

In an embodiment, the composition includes a compound of Formula 1 belowor a stereoisomer, a solvate, or a pharmaceutically acceptable saltthereof, wherein the drug is a physiologically active polypeptide:

X-L1-(OCH₂CH₂)_(n)O-L2-R  [Formula 1]

in Formula 1 above,

X is an immunoglobulin Fc region;

L1 is a straight or branched-chain C₁-C₆ alkylene;

L2 is -a1-CONH—, -a1-NHCO—, -a1-NHCO-a2-, —COO—, -b1-COO—, —COO-b2-, or-b1-COO-b2-, and at a2, b1, and b2 are each independently a straight orbranched-chain C₁-C₆ alkylene;

n is from 10 to 2400; and

R is any one selected from the group consisting of2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C₆-C₂₀aryl disulfide, C₅-C₂₀ heteroaryl disulfide, vinyl sulfone, thiol,halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester,and derivatives thereof.

In the composition according to the previous embodiment,

L1 is a straight or branched-chain C₁-C₆ alkylene;

L2 is -a1-NHCO— or -a1-NHCO-a2-;

a1 and a2 are each independently a straight or branched-chain C₁-C₆alkylene;

n is from 200 to 250; and

R is maleimide.

In the composition according to any of the previous embodiments, thephysiologically active polypeptide is selected from the group consistingof glucagon-like peptide-1 (GLP-1), granulocyte colony stimulatingfactor (G-CSF), human growth hormone (hGH), erythropoietin (EPO),glucagon, insulin, growth hormone releasing hormone, growth hormonereleasing peptide, interferons, interferon receptors, G-protein-coupledreceptors, interleukins, interleukin receptors, enzymes,interleukin-binding protein, cytokine-binding protein, macrophageactivating factor, macrophage peptide, B cell factor, T cell factor,protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin,lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growthfactor, α-1 antitrypsin, albumin, α-lactalbumin, apolipoprotein-E,highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin,thrombin receptor activating peptide, thrombomodulin, blood factors VII,Vila, VIII, IX, and XIII, plasminogen activating factor, fibrin-bindingpeptide, urokinase, streptokinase, hirudin, protein C, C-reactiveprotein, lenin inhibitor, collagenase inhibitor, superoxide dismutase,leptin, platelet-derived growth factor, epithelial growth factor,epidermal growth factor, angiostatin, angiotensin, bone growth factor,bone stimulating protein, calcitonin, atriopeptin, cartilage inducingfactor, elcatonin, connective tissue activating factor, tissue factorpathway inhibitor, follicle stimulating hormone, luteinizing hormone,luteinizing hormone releasing hormone, nerve growth factor, parathyroidhormone, relaxin, secretin, somatomedin, insulin-like growth factor,adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrinreleasing peptide, corticotropin releasing factor, thyroid stimulatinghormone, autotaxin, lactoferrin, myostatin, incretins, gastricinhibitory polypeptide (GIP), GLP-1/GIP dual agonist, GLP-1/GIP/Glucagontrigonal agonist, cell surface antigens, virus derived vaccine antigens,monoclonal antibodies, polyclonal antibodies, and antibody fragments.

In the composition according to any of the previous embodiments, R ofthe compound of the composition is linked to cysteine of the drug.

In the composition according to any of the previous embodiments, the Xis an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.

In the composition according to any of the previous embodiments, the Xis an immunoglobulin Fc region including a hinge sequence modified toinclude only one cysteine residue by deleting a part of an amino acidsequence of amino acids below:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

Still another aspect of the present invention provides a method forpreparing a long-acting conjugate of a physiologically activepolypeptide.

In an embodiment, the preparation method includes preparing a conjugateby linking a mono-PEGylated immunoglobulin Fc region, prepared bylinking a linker of Formula 3 below to the N-terminus of animmunoglobulin Fc region including a hinge sequence, to thephysiologically active polypeptide:

CHO-L1-(OCH₂CH₂)_(n)O-L2-R  [Formula 3]

wherein in Formula 3,

L1 is a straight or branched-chain C₁-C₆ alkylene;

L2 is -a1-CONH—, -a1-NHCO—, -a1-NHCO-a2-, —COO—, -b1-COO—, —COO-b2-, or-b1-COO-b2-, and at a2, b1, and b2 are each independently a straight orbranched-chain C₁-C₆ alkylene;

n is from 10 to 2400; and

R is any one selected from the group consisting of2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C₆-C₂₀aryl disulfide, C₅-C₂₀ heteroaryl disulfide, vinyl sulfone, thiol,halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester,and derivatives thereof.

In the preparation method according to the previous embodiment, themono-PEGylated immunoglobulin Fc region is prepared by linking thelinker of Formula 3 above to the N-terminus of the immunoglobulin Fcregion at a pH of 4.0 to 8.0 in the presence of a reducing agent.

In the preparation method according to any of the previous embodiments,the conjugate is prepared by linking the linker of the mono-PEGylatedimmunoglobulin Fc region to the physiologically active polypeptide at apH of 5.5 to 8.0.

In the preparation method according to any of the previous embodiments,the preparing of the conjugate is performed by reacting themono-PEGylated immunoglobulin Fc region with the physiologically activepolypeptide in a molar ratio of 1:1 to 1:3.

In the preparation according to any of the previous embodiments, themethod includes: preparing a mono-PEGylated immunoglobulin Fc region bylinking a linker of Formula 3 to the N-terminus of the immunoglobulin Fcregion; and preparing a conjugate by linking the linker of themono-PEGylated immunoglobulin Fc region prepared in the previous step toa physiologically active polypeptide.

In the preparation according to any of the previous embodiments, thelinker of the mono-PEGylated immunoglobulin Fc region is linked to acysteine of the physiologically active polypeptide.

In the preparation according to any of the previous embodiments, themethod includes: preparing a mono-PEGylated immunoglobulin Fc region bylinking a linker of Formula 3 to the N-terminus of an immunoglobulin Fcregion; purifying the mono-PEGylated immunoglobulin Fc region preparedin the previous step by anion-exchange chromatography in a buffersolution with a pH of 6.0 to 8.5; and preparing a conjugate by linkingthe linker of the mono-PEGylated immunoglobulin Fc region purified inthe previous step to a physiologically active polypeptide.

In the preparation according to any of the previous embodiments, themethod is performed without ultrafiltration/diafiltration afterpreparing the mono-PEGylated immunoglobulin Fc region.

In the preparation according to any of the previous embodiments, themethod further includes purifying the conjugate by hydrophobicinteraction chromatography.

In the preparation according to any of the previous embodiments, inFormula 3, L1 is a straight or branched-chain C₁-C₆ alkylene; L2 is-a1-NHCO— or -a1-NHCO— a2-; a1 and a2 are each independently a straightor branched-chain C₁-C₆ alkylene; n is from 200 to 250; and R ismaleimide.

In the preparation according to any of the previous embodiments, thelinker has a structure of Formula 4 below:

wherein in Formula 4, n is from 200 to 250.

In the preparation according to any of the previous embodiments, thelinker has a size of 1 kDa to 100 kDa.

In the preparation according to any of the previous embodiments, thephysiologically active polypeptide is selected from the group consistingof glucagon-like peptide-1 (GLP-1), granulocyte colony stimulatingfactor (G-CSF), human growth hormone (hGH), erythropoietin (EPO),glucagon, insulin, growth hormone releasing hormone, growth hormonereleasing peptide, interferons, interferon receptors, G-protein-coupledreceptors, interleukins, interleukin receptors, enzymes,interleukin-binding protein, cytokine-binding protein, macrophageactivating factor, macrophage peptide, B cell factor, T cell factor,protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin,lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growthfactor, α-1 antitrypsin, albumin, α-lactalbumin, apolipoprotein-E,highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin,thrombin receptor activating peptide, thrombomodulin, blood factors VII,Vila, VIII, IX, and XIII, plasminogen activating factor, fibrin-bindingpeptide, urokinase, streptokinase, hirudin, protein C, C-reactiveprotein, lenin inhibitor, collagenase inhibitor, superoxide dismutase,leptin, platelet-derived growth factor, epithelial growth factor,epidermal growth factor, angiostatin, angiotensin, bone growth factor,bone stimulating protein, calcitonin, atriopeptin, cartilage inducingfactor, elcatonin, connective tissue activating factor, tissue factorpathway inhibitor, follicle stimulating hormone, luteinizing hormone,luteinizing hormone releasing hormone, nerve growth factor, parathyroidhormone, relaxin, secretin, somatomedin, insulin-like growth factor,adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrinreleasing peptide, corticotropin releasing factor, thyroid stimulatinghormone, autotaxin, lactoferrin, myostatin, incretins, gastricinhibitory polypeptide (GIP), GLP-1/GIP dual agonist, GLP-1/GIP/Glucagontrigonal agonist, cell surface antigens, virus derived vaccine antigens,monoclonal antibodies, polyclonal antibodies, and antibody fragments.

In the preparation according to any of the previous embodiments, thephysiologically active polypeptide is a GLP-1/GIP/Glucagon trigonalagonist, glucagon, or an analog thereof.

In the preparation according to any of the previous embodiments, thephysiologically active polypeptide includes one of amino acid sequencesof SEQ ID NOS: 1 to 6.

In the preparation according to any of the previous embodiments, thehinge sequence is modified to include only one cysteine residue bydeleting a part of a hinge sequence having an amino acid sequence below:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

In the preparation according to any of the previous embodiments, thehinge sequence includes an amino acid sequence of SEQ ID NO: 8(Ser-Cys-Pro) or an amino acid sequence of SEQ ID NO: 9(Pro-Ser-Cys-Pro).

In the preparation according to any of the previous embodiments, theimmunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

Still another aspect of the present invention provides a long-actingdrug conjugate prepared using the composition or the method.

Advantageous Effects

According to the method for preparing a long-acting drug conjugate usinga novel intermediate according to the present invention, a long-actingdrug conjugate may be prepared with a high yield although some ofconventional purification processes are omitted, and thus productivityof the long-acting drug conjugate may be increased.

BRIEF DESCRIPTION OF DRAWINGS

FIGURE shows results of analyzing a structure of a mono-PEGylatedimmunoglobulin Fc region by MALDI-TOF assay.

BEST MODE

Hereinafter, embodiments of the present invention will be described indetail.

Meanwhile, each description and embodiment disclosed in the presentinvention may be applied herein to describe different descriptions andembodiments. In other words, all combinations of various componentsdisclosed in the present invention are included within the scope of thepresent invention. Furthermore, the scope of the present inventionshould not be limited by the detailed description provided below.

Also, those skilled in the art will recognize or be able to ascertain,using no more than routine experimentation, many equivalents to specificembodiments of the present invention. Such equivalents are intended tobe encompassed in the scope of the following claims.

Throughout the specification, not only the conventional one-letter andthree-letter codes for naturally occurring amino acids, but also thosethree-letter codes generally allowed for other amino acids, such asα-aminoisobutyric acid (Aib), N-methylglycine (Sar), andα-methyl-glutamic acid are used. In addition, the amino acids mentionedherein are abbreviated according to the nomenclature rules of IUPAC-IUBas follows.

alanine Ala, A

arginine Arg, R

asparagine Asn, N

aspartic acid Asp, D

cysteine Cys, C

glutamic acid Glu, E

glutamine Gln, Q

glycine Gly, G

histidine His, H

isoleucine Ile, I

leucine Leu, L

lysine Lys, K

methionine Met, M

phenylalanine Phe, F

proline Pro, P

serine Ser, S

threonine Thr, T

tryptophan Trp, W

tyrosine Tyr, Y

valine Val, V

An aspect of the present invention provides a compound having astructure of Formula 1 below, or a stereoisomer, a solvate, or apharmaceutically acceptable salt thereof:

X-L1-(OCH₂CH₂)_(n)O-L2-R  [Formula 1]

In Formula 1 above,

X is an immunoglobulin Fc region;

L1 is a straight or branched-chain C₁-C₆ alkylene;

L2 is -a1-CONH—, -a1-NHCO—, -a1-NHCO-a2-, —COO—, -b1-COO—, —COO-b2-, or-b1-COO-b2-, and a1, a2, b1, and b2 are each independently a straight orbranched-chain C₁-C₆ alkylene;

n is from 10 to 2400; and

R is any one selected from the group consisting of2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C₆-C₂₀aryl disulfide, C₅-C₂₀ heteroaryl disulfide, vinyl sulfone, thiol,halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester,and derivatives thereof.

In the present invention, the compound having a structure of Formula 1,or a stereoisomer, a solvate, or a pharmaceutically acceptable saltthereof is a novel substance prepared for preparing a long-actingconjugate and may also be referred to as “intermediate” or “intermediatematerial” in the present application.

In a method of preparing a long-acting drug conjugate using theintermediate of the present invention, purification steps byultrafiltration/diafiltration and hydrophobic interaction chromatographymay be omitted, and effects on preparing the long-acting drug conjugatewith a high yield may be obtained although the purification steps areomitted.

Specifically, the intermediate is in a form in which an immunoglobulinFc region is linked to a linker. In the intermediate of Formula 1, X maybe an immunoglobulin Fc region, and L1-(OCH₂CH₂)_(n)O-L2-R may be alinker. Specifically, in Formula 1 above, L1 is a straight orbranched-chain C₁-C₆ alkylene; L2 is -a1-NHCO— or -a1-NHCO-a2-; a1 anda2 are each independently a straight or branched-chain C₁-C₆ alkylene; nis from 200 to 250; and R is maleimide, without being limited thereto.

The L1 is a site binding to the immunoglobulin Fc region and may be astraight or branched-chain C₁-C₆ alkylene, without being limitedthereto. The L1 may be linked to an amine reactive group located at oneend or a lysine residue of X or thiol reactive group, without beinglimited thereto. The R is a site for linkage between the intermediateand a physiologically active polypeptide and, specifically, may includea reactive group (e.g., thiol, maleimide, aldehyde, and succinimidyl)capable of binding to a cysteine, or an amine group of the N-terminus,or a lysine residue of a physiologically active polypeptide, withoutbeing limited thereto.

In the present invention, the intermediate may have a structure ofFormula 2 below, but is not limited thereto:

In Formula 2 above, n is from 1 to 3000, from 10 to 2000, from 50 to1000, from 100 to 700, from 150 to 300, or from 200 to 250.

Specifically, the intermediate may have a size of 40 kDa to 250 kDa, 80kDa to 200 kDa, or 100 kDa to 150 kDa, without being limited thereto.

In the present invention, the X may be an immunoglobulin Fc regionhaving a hinge sequence at the N-terminus, and specifically, the hingesequence may be modified to include only one cysteine residue bydeleting a part of an amino acid sequence of the amino acid below,without being limited thereto:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

As used herein, the term “immunoglobulin Fc region” refers to a regionincluding a heavy chain constant domain 2 (CH2) and/or a heavy chainconstant domain 3 (CH3) excluding the heavy chain and light chainvariable domains of the immunoglobulin. The immunoglobulin Fc region maybe a component constituting a moiety of the long-acting drug conjugateof the present invention.

Specifically, the X may be an immunoglobulin Fc region derived from IgG,IgA, IgD, IgE, or IgM, more specifically, an immunoglobulin Fc regionderived from IgG1, IgG2, IgG3, or IgG4, without being limited thereto.

In the present invention, the immunoglobulin Fc region may include aparticular hinge sequence at the N-terminus.

As used herein, the term “hinge sequence” refers to a site located at aheavy chain and forming a dimer of the immunoglobulin Fc region via aninter disulfide bond.

As used herein, the term “N-terminus” refers to amino terminus of aprotein or polypeptide and may include an amino acid residue located atthe end of the amino terminus or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreamino acids from the end of the amino terminus. The immunoglobulin Fcregion of the present invention may include the hinge sequence at theN-terminus, without being limited thereto.

In view of the objects of the present invention, the hinge sequence mayinclude only one cysteine residue as a cysteine residue located at the8^(th) or 11^(th) position of the hinge sequence of SEQ ID NO: 7 isdeleted. The hinge sequence of the present invention may consist of 3 to12 amino acids including only one cysteine residue, without beinglimited thereto. More specifically, the hinge sequence of the presentinvention may have a sequence as follows:Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 11),Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 12),Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 13),Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 14),Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 15),Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 16),Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 17), Glu-Ser-Pro-Ser-Cys-Pro(SEQ ID NO: 18), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 19), Pro-Ser-Cys-Pro(SEQ ID NO: 20), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO:21), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 22),Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 23),Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 24),Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 25), Glu-Ser-Lys-Pro-Ser-Cys-Pro(SEQ ID NO: 26), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 27), orGlu-Pro-Ser-Cys (SEQ ID NO: 28). More specifically, the hinge sequencemay include an amino acid sequence of a sequence of SEQ ID NO: 8(Ser-Cys-Pro) or a sequence of SEQ ID NO: 9 (Pro-Ser-Cys-Pro), withoutbeing limited thereto.

The X may be a dimer formed of two chain molecules of the immunoglobulinFc region in the presence of the hinge sequence, and the intermediate ofthe present invention may be in a form in which one end of the linker islinked to one chain of the immunoglobulin Fc region in the dimeric form,without being limited thereto. In the present invention, the X may be adimer of the immunoglobulin Fc region, but is not limited thereto.

In addition, X of the present invention may be an immunoglobulin Fcregion including an amino acid sequence of SEQ ID NO: 10, without beinglimited thereto.

Meanwhile, the immunoglobulin Fc region of the present invention may bean extended Fc region including a part of or the entirety of a heavychain constant domain 1 (CH1) and/or a light chain constant domain 1(CL1) excluding the heavy chain and the light chain variable domains ofthe immunoglobulin, as long as the immunoglobulin Fc region hassubstantially identical or enhanced effects compared to the native type.Also, the immunoglobulin Fc region may be a region from which aconsiderably long part of the amino acid sequence corresponding to theCH2 and/or CH3 is removed.

For example, the immunoglobulin Fc region of the present invention mayinclude 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain andCH3 domain, 5) a combination of one or more domains selected from CH1domain, CH2 domain, CH3 domain, and CH4 domain and an immunoglobulinhinge region (or a part of the hinge region), or 6) a dimer of eachdomain of the heavy chain constant domain and the light chain constantdomain. However, the present invention is not limited thereto.

Also, the immunoglobulin Fc region of the present invention includes notonly a naturally occurring amino acid sequence but also a sequencederivative thereof. The amino acid sequence derivative refers to asequence different from the naturally occurring amino acid sequence dueto a deletion, insertion, non-conservative or conservative substitution,or any combination of one or more amino acids of the naturally occurringamino acid sequence.

For example, in the case of IgG Fc, amino acid residues known to beimportant in linkage at positions 214 to 238, 297 to 299, 318 to 322, or327 to 331 may be used as a suitable target for modification.

Also, other various derivatives including those in which a site capableof forming a disulfide bond is deleted or certain amino acid residuesare eliminated from the N-terminus of a native Fc form, and a methionineresidue is added to the N-terminus of the native Fc form may be used. Inaddition, to remove effector functions, a complement binding site, suchas a C1q binding site, may be deleted, and an antibody dependent cellmediated cytotoxicity (ADCC) site may be deleted. Techniques ofpreparing such sequence derivatives of the immunoglobulin Fc region aredisclosed in International Patent Publication Nos. WO 97/34631 and WO96/32478.

Amino acid exchanges in proteins and peptides, which do not generallyalter the activity of molecules, are known in the art (H. Neurath, R. L.Hill, The Proteins, Academic Press, New York, 1979). The most commonlyoccurring exchanges of amino acid residues are exchanges betweenAla/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val,Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu,and Asp/Gly. If required, the Fc region may be modified byphosphorylation, sulfation, acrylation, glycosylation, methylation,farnesylation, acetylation, and amidation.

The above-described sequence derivatives of the Fc region arederivatives that have a biological activity equivalent to that of theimmunoglobulin Fc region of the present invention or improved structuralstability against heat, pH, or the like.

In addition, these immunoglobulin Fc regions may be obtained from nativeforms isolated from humans and other animals including cows, goats,swine, mice, rabbits, hamsters, rats and guinea pigs, or may berecombinants or derivatives thereof, obtained from transformed animalcells or microorganisms. In this regard, they may be obtained from anative immunoglobulin by isolating whole immunoglobulins from livinghumans or animals and treating them with a protease. Papain digests thenative immunoglobulin into Fab and Fc regions and pepsin digests thenative immunoglobulin into pF′c and F(ab)₂ fragments. These fragmentsmay be subjected to size-exclusion chromatography to isolate Fc or pF′c.In a more specific embodiment, a human-derived Fc region is arecombinant immunoglobulin Fc region obtained from a microorganism.

In addition, the immunoglobulin Fc region of the present invention mayhave natural glycans or increased or decreased glycans compared to thenatural type, or be in a deglycosylated form. The increase, decrease, orremoval of glycans of the immunoglobulin Fc may be achieved by anymethods commonly used in the art such as a chemical method, an enzymaticmethod, and a genetic engineering method using a microorganism. In thisregard, the immunoglobulin Fc region obtained by removing glycans showsa significant decrease in binding affinity to a complement c1q and adecrease in or loss of antibody-dependent cytotoxicity orcomplement-dependent cytotoxicity, and thus unnecessary immune responsesare not induced thereby in living organisms. Based thereon, adeglycosylated or aglycosylated immunoglobulin Fc region may be moresuitable as a drug carrier in view of the objects of the presentinvention.

As used herein, the term “deglycosylation” refers to a Fc region fromwhich glycan is removed using an enzyme and the term “aglycosylation”refers to a Fc region that is not glycosylated and produced inprokaryotes, more specifically, E. coli.

Meanwhile, the immunoglobulin Fc region may be derived from humans oranimals such as cows, goats, swine, mice, rabbits, hamsters, rats, orguinea pigs. In a more specific embodiment, the immunoglobulin Fc regionmay be derived from humans.

In addition, the immunoglobulin Fc region may be derived from IgG, IgA,IgD, IgE, or IgM, or any combination or hybrid thereof. In a morespecific embodiment, the immunoglobulin Fc region is derived from IgG orIgM which are the most abundant proteins in human blood, and in an evenmore specific embodiment, it is derived from IgG known to enhance thehalf-lives of ligand-binding proteins. In a yet even more specificembodiment, the immunoglobulin Fc region is an IgG4 Fc region, and inthe most specific embodiment, the immunoglobulin Fc region is anaglycosylated Fc region derived from human IgG4, without being limitedthereto.

Meanwhile, as used herein, the term “combination” related to theimmunoglobulin Fc region refers to formation of a linkage between apolypeptide encoding a single-chain immunoglobulin Fc region of the sameorigin and a single-chain polypeptide of a different origin when a dimeror a multimer is formed. That is, a dimer or multimer may be preparedusing two or more Fc fragments selected from the group consisting of IgGFc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

As used herein, the term “hybrid” means that sequences corresponding totwo or more immunoglobulin Fc regions of different origins are presentin a single-chain of an immunoglobulin constant domain. In the presentinvention, various hybrid forms are possible. That is, a domain hybridmay be composed of 1 to 4 domains selected from the group consisting ofCH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc andmay further include a hinge region.

Meanwhile, IgG may also be classified into IgG1, IgG2, IgG3 and IgG4subclasses, which may be combined or hybridized in the presentinvention. Preferred are IgG2 and IgG4 subclasses, and most preferred isthe Fc fragment of IgG4 rarely having effector functions such ascomplement dependent cytotoxicity (CDC).

As used herein, the term “linker” refers to a moiety linking a drug(e.g., physiologically active polypeptide) to the immunoglobulin Fcregion in the long-acting drug conjugate, and the linker may be apeptidyl linker or a non-peptidyl linker. Specifically, the linker maybe represented by Formula 3 below, without being limited thereto:

CHO-L1-(OCH₂CH₂)_(n)O-L2-R  [Formula 3]

In Formula 3,

L1 is a straight or branched-chain C₁-C₆ alkylene;

L2 is -a1-CONH—, -a1-NHCO—, -a1-NHCO-a2-, —COO—, -b1-COO—, —COO-b2-, or-b1-COO-b2-, and a1, a2, b1, and b2 are each independently a straight orbranched-chain C₁-C₆ alkylene;

n is from 1 to 3000, from 10 to 2000, from 50 to 1000, from 100 to 700,from 150 to 300, or from 200 to 250; and

R is any one selected from the group consisting of2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C₆-C₂₀aryl disulfide, C₅-C₂₀ heteroaryl disulfide, vinyl sulfone, thiol,halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester,and derivatives thereof.

The linker may include polyethylene glycol and has particular chemicalstructures at both ends of polyethylene glycol, without being limitedthereto.

Specifically, in Formula 3 above, L1 may be a straight or branched-chainC₁-C₆ alkylene; L2 may be -a1-NHCO— or -a1-NHCO-a2-; a1 and a2 may beeach independently a straight or branched-chain C₁-C₆ alkylene; n may befrom 200 to 250; and R may be maleimide, and the linker may have a sizeof 1 kDa to 200 kDa, 1 kDa to 150 kDa, 1 kDa to 100 kDa, 1 kDa to 50kDa, or 1 kDa to 10 kDa, but is not limited thereto.

Also, the linker may have a structure of Formula 4 below, without beinglimited thereto:

In Formula 4 above, n may be from 1 to 3000, from 10 to 2000, from 50 to1000, from 100 to 700, from 150 to 300, or from 200 to 250.

One end of the linker may be linked to the immunoglobulin Fc region,specifically, the N-terminus of the immunoglobulin Fc region, morespecifically, the hinge sequence located at the N-terminus of theimmunoglobulin Fc region, even more specifically, a proline residue ofthe hinge sequence, to form the intermediate, but is not limitedthereto.

In the present invention, the term “pharmaceutically acceptable” refersto a substance that may be effectively used for the intended use withinthe scope of pharmaco-medical decision without inducing excessivetoxicity, irritation, or allergic responses.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt derived from a pharmaceutically acceptable inorganic acid, organicacid, or base. Examples of a suitable acid may include hydrochloricacid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaricacid, maleic acid, phosphoric acid, glycolic acid, lactic acid,salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid,acetic acid, citric acid, methanesulfonic acid, formic acid, benzoicacid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonicacid. Examples of the salt derived from a suitable base may includealkali metals such as sodium and potassium, alkali earth metals such asmagnesium, and ammonium.

The present invention includes not only the compound or apharmaceutically acceptable salt thereof, but also a solvate preparedtherefrom.

In addition, the compound may be present in the form of an enantiomer (Ror S isomer), racemate, or diastereomer, or any mixture thereof in thecase of having an asymmetric carbon center (absent carbon) in asubstituent thereof. In addition, the compound may be present in theform of an exo or endo isomer in the case of having a bridged ring,without being limited thereto.

An aspect of the present invention provides a composition including acompound having a structure of Formula 1 below, or a stereoisomer, asolvate, or a pharmaceutically acceptable salt thereof, wherein the drugis a physiologically active polypeptide:

X-L1-(OCH₂CH₂)_(n)O-L2-R  [Formula 1]

In Formula 1 above,

X is an immunoglobulin Fc region;

L1 is a straight or branched-chain C₁-C₆ alkylene;

L2 is -a1-CONH—, -a1-NHCO—, -a1-NHCO-a2-, —COO—, -b1-COO—, —COO-b2-, or-b1-COO-b2-, and a1, a2, b1, and b2 are each independently a straight orbranched-chain C₁-C₆ alkylene;

n is from 1 to 3000, from 10 to 2000, from 50 to 1000, from 100 to 700,from 150 to 300, or from 200 to 250; and

R is any one selected from the group consisting of2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C₆-C₂₀aryl disulfide, C₅-C₂₀ heteroaryl disulfide, vinyl sulfone, thiol,halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester,and derivatives thereof.

Specifically, in Formula 1 above, L1 is a straight or branched-chainC₁-C₆ alkylene; L2 is -a1-NHCO— or -a1-NHCO-a2-; a1 and a2 are eachindependently a straight or branched-chain C₁-C₆ alkylene; n is from 200to 250; and R is maleimide, without being limited thereto.

The composition of the present invention includes the intermediate andhas a use for preparing a long-acting drug conjugate.

Specifically, since the composition of the present invention includesthe intermediate in which the linker is linked to the immunoglobulin Fcregion, the composition of the present invention may be reacted with aphysiologically active polypeptide such that the linker of theintermediate is linked to the physiologically active polypeptide,thereby preparing a long-acting drug conjugate. More specifically, thelong-acting drug conjugate may be prepared via linkage between R ofFormula 1 corresponding to one end of the linker and a cysteine, or anamine group such as the N-terminus, or a lysine residue of thephysiologically active polypeptide, without being limited thereto.

In the present invention, any physiologically active polypeptide of thelong-acting drug conjugate that may be prepared using the compositionmay fall within the scope of the present invention regardless of type,size, origin, and the like as long as the physiologically activepolypeptide has pharmacological effects on disease. Examples of thephysiologically active polypeptide may include glucagon-like peptide-1(GLP-1), granulocyte colony stimulating factor (G-CSF), human growthhormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormonereleasing hormone, growth hormone releasing peptide, interferons,interferon receptors, G-protein-coupled receptors, interleukins,interleukin receptors, enzymes, interleukin-binding protein,cytokine-binding protein, macrophage activating factor, macrophagepeptide, B cell factor, T cell factor, protein A, allergy inhibitor,cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosisfactor, tumor suppressor, metastasis growth factor, α-1 antitrypsin,albumin, α-lactalbumin, apolipoprotein-E, highly glycosylatederythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptoractivating peptide, thrombomodulin, blood factors VII, Vila, VIII, IX,and XIII, plasminogen activating factor, fibrin-binding peptide,urokinase, streptokinase, hirudin, protein C, C-reactive protein, lenininhibitor, collagenase inhibitor, superoxide dismutase, leptin,platelet-derived growth factor, epithelial growth factor, epidermalgrowth factor, angiostatin, angiotensin, bone growth factor, bonestimulating protein, calcitonin, atriopeptin, cartilage inducing factor,elcatonin, connective tissue activating factor, tissue factor pathwayinhibitor, follicle stimulating hormone, luteinizing hormone,luteinizing hormone releasing hormone, nerve growth factor, parathyroidhormone, relaxin, secretin, somatomedin, insulin-like growth factor,adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrinreleasing peptide, corticotropin releasing factor, thyroid stimulatinghormone, autotaxin, lactoferrin, myostatin, incretins, gastricinhibitory polypeptide (GIP), GLP-1/GIP dual agonist, GLP-1/GIP/Glucagontrigonal agonist, cell surface antigens, virus derived vaccine antigens,monoclonal antibodies, polyclonal antibodies, and antibody fragments,but are not limited thereto.

More specifically, the physiologically active polypeptide may beglucagon-like peptide-1 (GLP-1), glucagon, insulin, enzyme, incretin,gastric inhibitory polypeptide (GIP), GLP-1/GIP dual agonist, orGLP-1/GIP/Glucagon triple agonist, but is not limited thereto.

Because a method for preparing a long-acting drug conjugate using theintermediate or the composition including the same according to thepresent invention is performed by linking the intermediate, in which thelinker is linked to the immunoglobulin Fc region, to the physiologicallyactive polypeptide, any physiologically active polypeptide including anamino acid residue or a reactive group capable of binding to theintermediate may be used regardless of types thereof to prepare thelong-acting drug conjugate using the intermediate or the compositionincluding the same of the present invention.

In the present invention, the X may be an immunoglobulin Fc regionderived from IgG1, IgG2, IgG3, or IgG4, without being limited thereto.

In addition, the X may be an immunoglobulin Fc region including a hingesequence modified to include only one cysteine residue by deleting apart of an amino acid sequence of amino acids below, but is not limitedthereto:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

In the case of using the composition of the present invention, thelong-acting drug conjugate may be prepared without performingultrafiltration/diafiltration and one cycle of hydrophobic interactionchromatography may be optionally omitted.

Specifically, when the long-acting drug conjugate is prepared byreacting the intermediate or the composition including the sameaccording to the present invention with the drug, purity of thelong-acting drug conjugate may be 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more, but is not limited thereto. Thepurity may be measured by any method well known in the art,specifically, by SE-HPLC, RP-HPLC, and IE-HPLC, but any method availablein the art. More specifically, the purity of the long-acting drugconjugate prepared using the composition of the present invention may be90% or more in SE-HPLC, 80% or more in RP-HPLC, and 85% or more inIE-HPLC, but is not limited thereto.

Also, the composition of the present invention may further include abuffer, a stabilizer, a preservative, a salt, and the like required tostabilize the intermediate and to prepare the long-acting drugconjugate, but is not limited thereto.

Another aspect of the present invention provides a kit for preparing along-acting drug conjugate including the composition. The kit mayinclude a reagent, manual and the like for preparing the long-actingdrug conjugate, without being limited thereto.

Another aspect of the present invention provides a method for preparinga long-acting drug conjugate.

The preparation method of the present invention is a method forpreparing a long-acting drug conjugate in which a drug is linked to animmunoglobulin Fc region via a linker, specifically, a method forpreparing a long-acting drug conjugate by linking the intermediate tothe drug, without being limited thereto.

Specifically, the preparation method is characterized by sequentiallyperforming i) linking a linker including polyethylene glycol (PEG) to animmunoglobulin Fc region, and ii) linking the linker, which is linked tothe immunoglobulin Fc region, to a drug (e.g., a physiologically activepolypeptide or protein). That is, the preparation method ischaracterized by performing steps in a particular order, i.e.,performing a first step of preparing the intermediate by linking thelinker including PEG to the immunoglobulin Fc region, and thenperforming a second step of linking the drug to the intermediate.Alternatively, the preparation method of the present invention may alsobe performed only by the second step of preparing the long-acting drugconjugate via a reaction between the intermediate or the composition forpreparing the long-acting drug conjugate including the same and thedrug, without performing the first step, but is not limited thereto.This preparation method may be referred to as “reverse order preparationmethod” in the present application.

In the present invention, in the case of the method for preparing thelong-acting drug conjugate performed by preparing the intermediate firstand then linking the intermediate to the drug, the purificationprocesses by ultrafiltration/diafiltration and hydrophobic interactionchromatography may be omitted and it was confirmed that the long-actingdrug conjugate may be prepared with a high yield although thepurification processes are omitted.

According to the convention preparation method in which the linker isfirst linked to the physiologically active polypeptide and then linkedto the immunoglobulin Fc region without forming the intermediate, whenthe physiologically active polypeptide-linked linker (e.g., polyethyleneglycol) is linked to the immunoglobulin Fc region,ultrafiltration/diafiltration is required as a separate process afterthe linker is linked to the physiologically active polypeptide andbefore the linked product is linked to the immunoglobulin Fc region toreduce the risk of aggregation that may occur due to low pH conditions(pH of about 3.0) of an equilibrium buffer and an elution buffer usedfor purification of the physiologically active polypeptide-linked linkerand to adjust the pH conditions for reaction using an appropriatebuffer. On the contrary, in the preparation method according to thepresent invention in which the intermediate is prepared by linking thelinker to the immunoglobulin Fc region first, a pH of a buffer used inpurification of the immunoglobulin Fc region-linked linker is relativelyhigh, and thus the ultrafiltration/diafiltration process may be omittedand then a process of linking the immunoglobulin Fc region-linked linkerto the physiologically active polypeptide may be performed.

Therefore, in the preparation method of the present invention,ultrafiltration/diafiltration may not be performed after preparing amono-PEGylated immunoglobulin Fc region, but the present invention isnot limited thereto. In the method for preparing a long-acting drugconjugate according to the present invention, a pH of a solution used topurify the mono-PEGylated immunoglobulin Fc region is not significantlydifferent from a pH of a solution used for a subsequent reaction so thatlinkage to the drug may be performed without conducting theultrafiltration/diafiltration. By omitting theultrafiltration/diafiltration process, the risk of formation ofaggregate impurities in a concentration step may be reduced and thepreparation process may be simplified so that cost reduction effects maybe expected in the case where the technology is commercialized.

Also, the preparation method of the present invention may furtherinclude purifying the conjugate by hydrophobic interactionchromatography, without being limited thereto.

Specifically, the hydrophobic interaction chromatography may beperformed only once or more than once in accordance with properties ofthe drug of the long-acting drug conjugate and type and size of thelinker.

According to the preparation method of the present invention, not onlyan amount of the expensive drug may be reduced but also an amount ofunreacted immunoglobulin Fc regions may be reduced, so that the entireor a part of the purification process by hydrophobic interactionchromatography may be omitted to obtain effects on reducing rawmaterials required for preparation of the long-acting drug conjugate andcosts therefor compared to the conventional method.

Meanwhile, although the ultrafiltration/diafiltration and hydrophobicinteraction chromatography processes, which have been performed in theconventional method for preparing the long-acting drug conjugate, areomitted and only the final purification process (e.g., one cycle ofhydrophobic interaction chromatography) is performed in the preparationmethod of the present invention, it is advantageous in that a purity ofthe final conjugate obtained by the present invention is maintainedcompared to that of the conventional preparation method. That is,according to the preparation method of the present invention, the finalpurity may be maintained although some of the purification processes areomitted so that productivity of the long-acting drug conjugate may beimproved.

The purity of the long-acting drug conjugate according to the presentinvention may be measured by any method well known in the art andexamples of the method may be SE-HPLC, RP-HPLC, and IE-HPLC, withoutbeing limited thereto.

According to the preparation method of the present invention, the finalpurity of the long-acting drug conjugate may be 90% or more,specifically, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more,without being limited thereto.

Meanwhile, in the preparation method of the present invention, themono-PEGylated immunoglobulin Fc region is prepared first and thenlinked to the physiologically active polypeptide, so that thelong-acting drug conjugate may be prepared with a higher yield comparedto the conventional method in terms of not only the physiologicallyactive polypeptide but also the immunoglobulin Fc region.

In an embodiment of the present invention, it was confirmed that theyield of the long-acting drug conjugate obtained by the preparationmethod of the present invention was increased twice or more compared tothe yield of the long-acting drug conjugate obtained by the conventionalmethod.

Specifically, the preparation method of the present invention relates toa method for preparing a long-acting drug conjugate including preparinga conjugate by linking a mono-PEGylated immunoglobulin Fc region, whichis prepared by linking a linker of Formula 3 below to the N-terminus ofan immunoglobulin Fc region including a hinge sequence, to a drug(physiologically active polypeptide:

CHO-L1-(OCH₂CH₂)_(n)O-L2-R  [Formula 3]

In Formula 3 above,

L1 is a straight or branched-chain C₁-C₆ alkylene;

L2 is -a1-CONH—, -a1-NHCO—, -a1-NHCO-a2-, —COO—, -b1-COO—, —COO-b2-, or-b1-COO-b2-, and at a2, b1, and b2 are each independently a straight orbranched-chain C₁-C₆ alkylene;

n is from 10 to 2400; and

R is any one selected from the group consisting of2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C₆-C₂₀aryl disulfide, C₅-C₂₀ heteroaryl disulfide, vinyl sulfone, thiol,halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester,and derivatives thereof, without being limited thereto.

More specifically,

the preparation method may include: preparing a mono-PEGylatedimmunoglobulin Fc region by linking a linker of Formula 3 above to theN-terminus of an immunoglobulin Fc region; and preparing a conjugate bylinking the linker of the mono-PEGylated immunoglobulin Fc regionprepared in the above-described step to a physiologically activepolypeptide, or

the preparation method may include: preparing a mono-PEGylatedimmunoglobulin Fc region by linking a linker of Formula 3 to theN-terminus of an immunoglobulin Fc region; purifying the mono-PEGylatedimmunoglobulin Fc region prepared in the above-described step byanion-exchange chromatography in a buffer solution with a pH of 6.0 to8.5, a pH of 6.0 to 8.0, a pH of 6.0 to 7.5, a pH of 6.0 to 7.0, a pH of6.1 to 6.9, a pH of 6.2 to 6.8, or a pH of 6.3 to 6.7; and preparing aconjugate by linking the linker of the mono-PEGylated immunoglobulin Fcregion purified in the above-described step to a physiologically activepolypeptide, without being limited thereto.

In addition, in the preparation method of the present invention,

(i) the mono-PEGylated immunoglobulin Fc region may be prepared bylinking the linker of Formula 3 to the N-terminus of the immunoglobulinFc region in the presence of a reducing agent at a pH of 4.0 to 8.0, apH of 4.5 to 7.5, a pH of 5.5 to 7.5, a pH of 5.6 to 7.4, a pH of 5.7 to7.3 or a pH of 5.8 to 7.2; and/or

(ii) the conjugate may be prepared by linking the linker of themono-PEGylated immunoglobulin Fc region to the physiologically activepolypeptide at a pH of 5.5 to 8.0, a pH of 6.0 to 7.5, or a pH of 6.5 to7.5, without being limited thereto.

In addition, the step of preparing the conjugate according to thepreparation method of the present invention may be performed by reactingthe physiologically active polypeptide in an amount equivalent to ormore than an amount of the mono-PEGylated immunoglobulin Fc region, andspecifically, a molar ratio of mono-PEGylated immunoglobulin Fc region:physiologically active polypeptide may be from 1:1 to 1:10, from 1:1 to1:7, from 1:1 to 1:5, or from 1:1 to 1:3, but is not limited thereto.

As used herein, the term “mono-PEGylated immunoglobulin Fc region”refers to an intermediate material that is produced in the middle of themethod for preparing the long-acting drug conjugate according to thepresent invention in which one linker including one polyethylene glycolis linked to the immunoglobulin Fc region. That is, in the presentinvention, the “mono-PEGylated immunoglobulin Fc region” may be usedinterchangeably with “intermediate” or “intermediate material”.

In the present invention, the immunoglobulin Fc region may be animmunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4, withoutbeing limited thereto.

In addition, the immunoglobulin Fc region may be an immunoglobulin Fcregion including a hinge sequence modified to include only one cysteineresidue by deleting a part of an amino acid sequence below, andspecifically, the hinge sequence may include an amino acid sequence ofSEQ ID NO: 8 (Ser-Cys-Pro) or SEQ ID NO: 9 (Pro-Ser-Cys-Pro), withoutbeing limited thereto.

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

The “long-acting drug conjugate” of the present invention refers to adrug conjugate having a structure in which a drug (physiologicallyactive polypeptide) having a pharmacological activity in the body islinked to an immunoglobulin Fc region via a linker and an increasedhalf-life. In view of the objects of the present invention, thelong-acting drug conjugate may be one in which the intermediate ormono-PEGylated immunoglobulin Fc region is linked to the drug, but isnot limited thereto.

Specifically, the drug is not limited to particular substances as longas the drug has preventive, therapeutic, or alleviating effects on acertain disease and may be a natural or non-natural protein, enzyme,antibody, compound, or the likes. More specifically, the drug may be aphysiologically active polypeptide or protein, even more specifically,the physiologically active polypeptide may be glucagon-like peptide-1(GLP-1), granulocyte colony stimulating factor (G-CSF), human growthhormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormonereleasing hormone, growth hormone releasing peptide, interferons,interferon receptors, G-protein-coupled receptors, interleukins,interleukin receptors, enzymes, interleukin-binding protein,cytokine-binding protein, macrophage activating factor, macrophagepeptide, B cell factor, T cell factor, protein A, allergy inhibitor,cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosisfactor, tumor suppressor, metastasis growth factor, α-1 antitrypsin,albumin, α-lactalbumin, apolipoprotein-E, highly glycosylatederythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptoractivating peptide, thrombomodulin, blood factors VII, Vila, VIII, IX,and XIII, plasminogen activating factor, fibrin-binding peptide,urokinase, streptokinase, hirudin, protein C, C-reactive protein, lenininhibitor, collagenase inhibitor, superoxide dismutase, leptin,platelet-derived growth factor, epithelial growth factor, epidermalgrowth factor, angiostatin, angiotensin, bone growth factor, bonestimulating protein, calcitonin, atriopeptin, cartilage inducing factor,elcatonin, connective tissue activating factor, tissue factor pathwayinhibitor, follicle stimulating hormone, luteinizing hormone,luteinizing hormone releasing hormone, nerve growth factor, parathyroidhormone, relaxin, secretin, somatomedin, insulin-like growth factor,adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrinreleasing peptide, corticotropin releasing factor, thyroid stimulatinghormone, autotaxin, lactoferrin, myostatin, incretins, gastricinhibitory polypeptide (GIP), GLP-1/GIP dual agonist, GLP-1/GIP/Glucagontrigonal agonist, cell surface antigens, virus derived vaccine antigens,monoclonal antibodies, polyclonal antibodies, and antibody fragments,but are not limited thereto. More specifically, the physiologicallyactive polypeptide may be GLP-1/GIP/Glucagon trigonal agonist, glucagon,or a analog thereof, but is not limited thereto. Even more specifically,the physiologically active polypeptide may include, essentially consistof, or consist of one of amino acid sequences of SEQ ID NOS: 1 to 6, butis not limited thereto.

In addition, any variants, derivatives, and fragments of thephysiologically active polypeptide also fall within the scope of thepresent invention.

As used herein, the term “variant” refers to a peptide having an aminoacid sequence in which one or more amino acids are different from thoseof a native physiologically active polypeptide while retaining the samefunctions as those of the native physiologically active polypeptide, andthe variant may be prepared by substitution, addition, deletion,modification, or any combination of some amino acids of the amino acidsequence of the native physiologically active polypeptide.

As used herein, the term “derivative” refers to a peptide, a peptideanalog, or a peptidomimetic obtained by modifying one or more aminoacids of the native physiologically active polypeptide by addition,deletion, or substitution to have similar activity to that of the nativephysiologically active polypeptide.

As used herein, the term “fragment” refers to a form obtained byadding/deleting one or more amino acids to/from the N-terminus or theC-terminus, and the added amino acid may be any amino acid that does notexist in nature (e.g.; D-amino acid).

The methods for preparing the variant, derivative, and fragment of thephysiologically active polypeptide may be used independently or incombination. For example, any physiologically active polypeptide havingone or more different amino acids in the amino acid sequence anddeamination of an amino acid residue at the N-terminus may be includedtherein.

The derivative of the physiologically active polypeptide includesbiosimilar and biobetter forms. For example, with respect tobiosimilars, the biosimilar may be any biosimilar enzyme available inthe long-acting drug conjugate of the present invention although thereis a difference between a known enzyme and a host for its expression, adifference in glycosylation feature and the degree thereof, and adifference in the degree of substitution in a particular amino acidresidue of the corresponding enzyme in light of the standard sequencewhere the degree of substitution is not 100% substitution. Thephysiologically active polypeptide and the variant, derivative andfragment thereof may be produced from animal cells, E. coli, yeast,insect cells, plant cells, living animals, and the like via geneticrecombination, the production methods are not limited thereto, and anycommercially available physiologically active polypeptides, andvariants, derivatives, and fragments thereof may also be used.

In addition, the physiologically active polypeptide, and the variant,derivative and fragment thereof may include an amino acid sequencehaving a homology of at least 80%, specifically, at least 90%, morespecifically, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,and the physiologically active polypeptide, and the variant, derivative,and fragment thereof may be obtained from microorganisms by geneticrecombination technologies or commercially available, without beinglimited thereto.

As used herein, the term “homology” refers to the degree of similaritybetween amino acid sequences of a wild-type protein or nucleotidesequences encoding the same and includes a sequence identical to theamino acid sequence or nucleotide sequence of the present invention bythe above-described percentage or more. The homology may be determinedby comparing the sequences via visual observation but may also bedetermined using a bioinformatic algorithm, which provides analysisresults of a degree of homology by aligning sequences to be compared.The homology between the two amino acid sequences may be indicated inpercentage. Useful automated algorithms may be used in GAP, BESTFIT,FASTA, and TFASTA computer software modules of Wisconsin GeneticsSoftware Package (Genetics Computer Group, Madison, Wis., USA). Theautomated alignment algorithms in the modules include the Needleman &Wunsch algorithm, the Pearson & Lipman algorithm, and the Smith &Waterman sequence algorithm. Other useful algorithms and homologydeterminations on alignment are automated in software such as FASTP,BLAST, BLAST2, PSIBLAST, and CLUSTAL W.

Information on sequences of the physiologically active polypeptide, andthe variant, derivative, and fragment thereof, and nucleotide sequencesencoding the same may be obtained from known database of the NCBIGenBank, or the like.

The amino acids substituted or added may be not only 20 amino acidscommonly found in human proteins but also atypical or non-naturallyoccurring amino acids. Commercial sources of atypical amino acids mayinclude Sigma-Aldrich, Chem Pep Inc. and Genzyme pharmaceuticals. Thepeptides including theses amino acids and typical peptide sequences maybe synthesized and purchased from commercial suppliers, e.g., AmericanPeptide Company, Bachem (USA) or Anygen (Korea).

In addition, the physiologically active polypeptide, and the variant,derivative, and fragment thereof according to the present invention maybe in a varied form where the N-terminus and/or C-terminus is chemicallymodified or protected by organic groups, or amino acids may be added tothe termini of the peptide, for protection from proteases in vivo whileincreasing stability thereof.

Particularly, since the N- and C-termini of chemically-synthesizedpeptides are electrically charged, the N-terminus may be acetylatedand/or the C-terminus may be amidated to remove the charges, but theembodiment is not limited thereto.

In addition, the peptide according to the present invention includes allof those in the form of the peptide itself, a salt thereof (e.g., apharmaceutically acceptable salt of the peptide), or a solvate thereof.Also, the peptide may be in any pharmaceutically acceptable form.

The type of the salt is not particularly limited. However, the salt ispreferably in a form safe and effective to an individual, e.g., amammal, without being limited thereto.

As used herein, the term “solvate” refers to a complex of the peptide ora salt thereof according to the present invention and a solventmolecule.

Although described as a “peptide consisting of a particular SEQ ID NO”in the present invention, it does not exclude a mutation that may occurnaturally or by addition of a meaningless sequence upstream ordownstream of the amino acid sequence of the SEQ ID NO, or a silentmutation thereof, as long as the peptide has activity identical orequivalent to that of the peptide consisting of the amino acid sequence,and even when such sequence addition or mutation is present, itobviously belongs to the scope of the present invention.

The method for preparing the long-acting drug conjugate according to thepresent invention may be a method for preparing a conjugate in which aphysiologically active polypeptide is linked to an immunoglobulin Fcregion via a linker, without being limited thereto.

In the present invention, linkage between the linker and theimmunoglobulin Fc region may be formed by a covalent bond or anon-covalent bond between one end of the linker and the N-terminus ofthe immunoglobulin Fc region, but binding sites or methods for thelinkage are not particularly limited. Specifically, the mono-PEGylatedimmunoglobulin Fc region may be prepared by linking a proline at theN-terminus of the immunoglobulin Fc region to a —CHO group of thelinker, without being limited thereto.

In the present invention, the linker may have a structure of Formula 4below, but is not limited thereto:

In Formula 4 above, n is from 200 to 250.

In addition, the linker may have a size of 1 kDa to 200 kDa, 1 kDa to150 kDa, 1 kDa to 100 kDa, 1 kDa to 50 kDa, or 1 kDa to 10 kDa, withoutbeing limited thereto.

The mono-PEGylated immunoglobulin Fc region may have a structure ofFormula 2 below, without being limited thereto.

The preparation method of the present invention may be performed bylinking the physiologically active polypeptide to one end of themono-PEGylated immunoglobulin Fc region having the structure of Formula2, without being limited thereto.

In addition, the other end of the linker which is not linked to theimmunoglobulin Fc region may be linked to the physiologically activepolypeptide, specifically, a —SH group or an amino acid containing a —SHgroup, or a cysteine of the physiologically active polypeptide, withoutbeing limited thereto.

In the present invention, the long-acting drug conjugate may have astructure of Formula 5 below, without being limited thereto.

Formula 5 shows a structure in which the physiologically activepolypeptide, the linker, and the immunoglobulin Fc region aresequentially linked from the left, without being limited thereto.

When the long-acting drug conjugate is prepared according to thepreparation method of the present invention in which the mono-PEGylatedimmunoglobulin Fc region is prepared first and linked to thephysiologically active polypeptide, it was confirmed that the purity ofthe final conjugate may be maintained with an increased yield comparedto the conventional preparation method although theultrafiltration/diafiltration and hydrophobic interaction chromatographyprocesses are omitted and only the final purification process (e.g., onecycle of hydrophobic interaction chromatography) is performed in thepreparation method according to the present invention.

Another aspect of the present invention provides a long-acting drugconjugate prepared by the above-described method.

Because the long-acting drug conjugate prepared by the preparation ofthe present invention has an increased half-life compared to thephysiologically active polypeptide that is not linked to the linker orthe immunoglobulin Fc region, advantageous effects on preparation ofdrugs may be obtained.

The long-acting drug conjugate prepared by the preparation method of thepresent invention may be used in preparation of drugs or compositionsfor the purposes of prevention, treatment, and alleviation of diseases.

Hereinafter, the present invention will be described in more detail withreference to the following examples. However, the following examples aremerely presented to exemplify the present invention, and the scope ofthe present invention is not limited thereto.

Comparative Example: Preparation of Conjugate by Linking PEGylatedPhysiologically Active Polypeptide to Immunoglobulin Fc Region

A PEGylated physiologically active polypeptide was linked to animmunoglobulin Fc region to prepare a long-acting conjugate.

Comparative Example 1: Preparation of Conjugate by Linking PEGylatedGLP-1/GIP/Glucagon Trigonal Agonist Analog 1 to Immunoglobulin Fc Region

In order to PEGylate a physiologically active polypeptide(GLP-1/GIP/Glucagon trigonal agonist analog 1, SEQ ID NO: 1) at acysteine residue (—SH group), the GLP-1/GIP/Glucagon trigonal agonistanalog 1 was reacted with a linker containing PEG (maleimide-10kDa-PEG-aldehyde) (Formula 4) for about 1 hour in a molar ratio of 1:1.0to 1:1.3 with a GLP-1/GIP/Glucagon trigonal agonist analog 1concentration of about 3 g/L. Specifically, the reaction was performedin a 50 mM Tris buffer containing isopropanol (pH of 7.5, 6° C.±4° C.).In order to obtain a mono-PEGylated GLP-1/GIP/Glucagon trigonal agonistanalog 1, the reaction solution was diluted with an equilibrium bufferincluding sodium citrate and ethanol to a total volume of 20 times andpurified. In this regard, the mono-PEGylated GLP-1/GIP/Glucagon trigonalagonist analog 1 was purified using an SP High Performance column (GEHealthcare, cation-exchange chromatography) using a solution includingsodium citrate and ethanol and a potassium chloride concentrationgradient. After the purified solution of the PEGylatedGLP-1/GIP/Glucagon trigonal agonist analog 1 was diluted with water, thebuffer solution was replaced with a 0.1 M potassium phosphate solutionthrough ultrafiltration/diafiltration (UF/DF), followed by concentrationto recover a resultant with a final concentration of about 3 g/L ormore.

The mono-PEGylated GLP-1/GIP/Glucagon trigonal agonist analog 1 preparedas described above was linked to an immunoglobulin Fc region to preparea long-acting conjugate as follows.

In order to link an aldehyde group of PEG of the mono-PEGylatedGLP-1/GIP/Glucagon trigonal agonist analog 1 to the amino terminus of animmunoglobulin Fc region, the mono-PEGylated GLP-1/GIP/Glucagon trigonalagonist analog 1 was reacted with the immunoglobulin Fc region in amolar ratio of 1:2 at a temperature of 6° C.±4° C. for about 12 hourswith a total protein concentration (GLP-1/GIP/Glucagon trigonal agonistanalog 1 and immunoglobulin Fc region) of 30 g/L.

In order to isolate and remove unreacted immunoglobulin Fc regions afterthe reaction for linkage, the reaction solution was purified using aButyl 4 Fast Flow column (GE Healthcare, hydrophobic interactionchromatography). In this case, a Tris buffer and sodium chloride wereadded to the reaction solution, and the reaction solution was purifiedusing a solution including a Bis-Tris and a sodium chlorideconcentration gradient.

Thereafter, using a Source 15ISO column (GE Healthcare), hydrophobicinteraction chromatography was performed. By-products were eliminated bythis process, and an immunoglobulin Fc region-PEG-containinglinker-GLP-1/GIP/Glucagon trigonal agonist analog 1 conjugate wasobtained. In this case, purification was performed using a bufferincluding sodium citrate and an ammonium sulfate concentration gradient.

Comparative Example 2: Preparation of Conjugate by Linking PEGylatedGlucagon Analog 1 to Immunoglobulin Fc Region

In order to PEGylate a physiologically active polypeptide (Glucagonanalog 1, SEQ ID NO: 4) at a cysteine residue (—SH group), Glucagonanalog 1 was reacted with a linker containing PEG (maleimide-10kDa-PEG-aldehyde) (Formula 1) for about 1 hour in a molar ratio of 1:1.3with a Glucagon analog 1 concentration of 3 g/L. Specifically, thereaction was performed in a 50 mM Tris buffer containing isopropanol (pHof 7.3). In order to obtain a mono-PEGylated Glucagon analog 1, thereaction solution was diluted with an equilibrium buffer includingsodium citrate and ethanol to a total volume of 20 times and purified.In this regard, the mono-PEGylated Glucagon analog 1 was purified usingan SP High Performance column (GE Healthcare, cation-exchangechromatography) using a solution including sodium citrate and ethanoland a potassium chloride concentration gradient. After the purifiedsolution of the PEGylated Glucagon analog 1 was diluted with water, thebuffer solution was replaced with a 0.1 M potassium phosphate solutionthrough ultrafiltration/diafiltration (UF/DF), followed by concentrationto recover a resultant with a final concentration of 3 g/L or more.

The mono-PEGylated Glucagon analog 1 prepared as described above waslinked to an immunoglobulin Fc region to prepare a long-acting conjugateas follows.

In order to link an aldehyde group of PEG of the mono-PEGylated Glucagonanalog 1 to the amino terminus of the immunoglobulin Fc region, themono-PEGylated Glucagon analog 1 was reacted with the immunoglobulin Fcregion in a molar ratio of 1:5 at a temperature of 6° C.±4° C. for about12 hours with a total protein concentration (Glucagon analog 1 andimmunoglobulin Fc region) of 20 g/L.

In order to isolate and remove unreacted immunoglobulin Fc regions afterthe reaction for linkage, the reaction solution was purified using aButyl 4 Fast Flow column (GE Healthcare, hydrophobic interactionchromatography). In this case, a Tris buffer and sodium chloride wereadded to the reaction solution, and the reaction solution was purifiedusing a solution including Bis-Tris and a sodium chloride concentrationgradient.

Thereafter, using a Source 15ISO column (GE Healthcare), hydrophobicinteraction chromatography was performed. By-products were eliminated bythis process, and an immunoglobulin Fc region-PEG-containinglinker-Glucagon analog 1 conjugate was obtained. In this case,purification was performed using a buffer including sodium citrate andan ammonium sulfate concentration gradient.

The present inventors have developed a process capable of efficientlyproducing the conjugate with a high purity by omitting the membranefiltration process and the purification process (hydrophobic interactionchromatography, Butyl 4 Fast Flow) from the process of preparing theconjugate according to the above-described Comparative Examples 1 and 2as follows.

Example 1: Preparation of Mono-PEGylated Immunoglobulin Fc RegionExample 1-1. Preparation of Mono-PEGylated Immunoglobulin Fc Region

In order to PEGylate the N-terminus of an immunoglobulin Fc region (49.8kDa) having a hinge region with a Pro-Ser-Cys-Pro (SEQ ID NO: 9)sequence at the N-terminus, the immunoglobulin Fc region was reactedwith a linker containing PEG (structure of Formula 4, 10 kDa) in a molarratio (immunoglobulin Fc region: PEG-containing linker) of 1:1 with animmunoglobulin Fc region concentration of 50 g/L at 6° C.±4° C. forabout 4 hours.

Specifically, the reaction was performed in a composition including a 5mM Bis-Tris buffer (pH 6.5) and potassium phosphate, and 10 mM NaCNBH₃(sodium cyanoborohydride) was added thereto as a reducing agent. Inorder to obtain a mono-PEGylated immunoglobulin Fc region, the reactionsolution was diluted with the Bis-Tris buffer and purified.

Unlike the preparation method of the above-described ComparativeExamples in which the mono-PEGylated GLP-1/GIP/Glucagon trigonal agonistanalog and glucagon analog were purified by cation-exchangechromatography, the mono-PEGylated immunoglobulin Fc region was purifiedusing a CaptoQ ImpRes column (GE Healthcare, anion-exchangechromatography) using a Bis-Tris buffer and a sodium chlorideconcentration gradient.

Example 1-2. Analysis of Structure of Mono-PEGylated Immunoglobulin FcRegion

The mono-PEGylated immunoglobulin Fc region prepared in Example 1-1 wasstructurally analyzed by MALDI-TOF and Peptide mapping. As a result ofMALDI-TOF, the resultant was identical to an expected molecular weightof the mono-PEGylated immunoglobulin Fc region (FIG. 1 ), and as aresult of Peptide mapping, it was confirmed that over 90% of PEG wasPEGylated at the N-terminus of the immunoglobulin Fc region.

Meanwhile, as a result of analyzing the mono-PEGylated immunoglobulin Fcregion (Formula 2) prepared in Example 1-1 above using SE-HPLC, RP-HPLC,and IE-HPLC assays, the purity was confirmed to be 90% or more inSE-HPLC, 90% or more in RP-HPLC, and 80% or more in IE-HPLC.

Example 2: Preparation of Conjugate by Linking PEGylated ImmunoglobulinFc Region to Physiologically Active Polypeptide

Long-acting conjugates were prepared as follows by linking themono-PEGylated immunoglobulin Fc region prepared in Example 1-1 tovarious physiologically active peptides.

Unlike the preparation method of the Comparative Examples where thePEGylated physiologically active polypeptide was purified bycation-exchange chromatography and then subjected to buffer exchange andconcentration by ultrafiltration/diafiltration (UF/DF), themono-PEGylated immunoglobulin Fc region was reacted with thephysiologically active polypeptide via peptide conjugation withoutperforming ultrafiltration/diafiltration. Long-acting conjugatesprepared as described above had high purity, and thus one of the twocycles of hydrophobic interaction chromatography could be omitted unlikethe preparation method according to the Comparative Examples.

Example 2-1. Preparation of Conjugate of GLP-1/GIP/Glucagon TrigonalAgonist Analog 1

A long-acting conjugate (immunoglobulin Fc region-PEG-containinglinker-GLP-1/GIP/Glucagon trigonal agonist analog 1) was prepared viapeptide conjugation of the GLP-1/GIP/Glucagon trigonal agonist analog 1(SEQ ID NO: 1), after anion-exchange chromatography of Example 1-1,without performing ultrafiltration/diafiltration.

In this regard, in order to link a maleimide reactive group at oneterminus of PEG of the mono-PEGylated immunoglobulin Fc region to theGLP-1/GIP/Glucagon trigonal agonist analog 1, the mono-PEGylatedimmunoglobulin Fc region was reacted with the GLP-1/GIP/Glucagontrigonal agonist analog 1 in a molar ratio of 1:1 with aGLP-1/GIP/Glucagon trigonal agonist analog 1 concentration of 0.2 g/L at6° C.±4° C. for about 2 hours. The reaction was performed in a Tris-Clbuffer (6° C.±4° C.) including isopropanol. As a result of analyzing theresultant after reaction using SE-HPLC, RP-HPLC, and IE-HPLC assays, thepurity was confirmed to be 90% or more in SE-HPLC, 80% or more inRP-HPLC, and 70% or more in IE-HPLC.

Thereafter, the resultant of the reaction was subjected to hydrophobicinteraction chromatography once using a Source 15ISO column (GEHealthcare). By-products were eliminated by this process, and animmunoglobulin Fc region-PEG-containing linker-GLP-1/GIP/Glucagontrigonal agonist analog 1 conjugate was obtained. In this case,purification was performed using a buffer including sodium citrate andan ammonium sulfate concentration gradient. It was confirmed that ayield obtained herein was increased by about twice or more compared to ayield of Comparative Example 1 with the same amount of theGLP-1/GIP/Glucagon trigonal agonist analog 1.

The eluted immunoglobulin Fc region-PEG-containinglinker-GLP-1/GIP/Glucagon trigonal agonist analog 1 conjugate wasanalyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity wasconfirmed since the purity was 90% or more in SE-HPLC, 90% or more inRP-HPLC, and 90% or more in IE-HPLC.

Example 2-2. Preparation of Conjugate of GLP-1/GIP/Glucagon TrigonalAgonist Analog 2

A long-acting conjugate (immunoglobulin Fc region-PEG-containinglinker-GLP-1/GIP/Glucagon trigonal agonist analog 2) was prepared viapeptide conjugation of the GLP-1/GIP/Glucagon trigonal agonist analog 2(SEQ ID NO: 2), after anion-exchange chromatography of Example 1-1,without performing ultrafiltration/diafiltration.

In this regard, in order to link a maleimide reactive group at oneterminus of PEG of the mono-PEGylated immunoglobulin Fc region to theGLP-1/GIP/Glucagon trigonal agonist analog 2, the mono-PEGylatedimmunoglobulin Fc region was reacted with the GLP-1/GIP/Glucagontrigonal agonist analog 2 in a molar ratio of 1:1 with aGLP-1/GIP/Glucagon trigonal agonist analog 2 concentration of 0.2 g/L at6° C.±4° C. for about 2 hours. The reaction was performed in a Tris-Clbuffer (6° C.±4° C.) including isopropanol. As a result of analyzing theresultant after reaction using SE-HPLC, RP-HPLC, and IE-HPLC assays, thepurity of the long-acting conjugate including the GLP-1/GIP/Glucagontrigonal agonist analog 2 was confirmed to be 90% or more in SE-HPLC,80% or more in RP-HPLC, and 70% or more in IE-HPLC.

Thereafter, the resultant of the reaction was subjected to hydrophobicinteraction chromatography once using a Source 15ISO column (GEHealthcare). By-products were eliminated by this process, and animmunoglobulin Fc region-PEG-containing linker-GLP-1/GIP/Glucagontrigonal agonist analog 2 conjugate was obtained. In this case,purification was performed using a buffer including sodium citrate andan ammonium sulfate concentration gradient. It was confirmed that ayield obtained herein was increased by about twice or more compared to ayield of Comparative Example 1 with the same amount of theGLP-1/GIP/Glucagon trigonal agonist analog 2.

The eluted immunoglobulin Fc region-PEG-containinglinker-GLP-1/GIP/Glucagon trigonal agonist analog 2 conjugate wasanalyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity wasconfirmed since the purity was 90% or more in SE-HPLC, 90% or more inRP-HPLC, and 80% or more in IE-HPLC.

Example 2-3. Preparation of Conjugate of GLP-1/GIP/Glucagon TrigonalAgonist Analog 3

A long-acting conjugate (immunoglobulin Fc region-PEG-containinglinker-GLP-1/GIP/Glucagon trigonal agonist analog 3) was prepared viapeptide conjugation of the GLP-1/GIP/Glucagon trigonal agonist analog 3(SEQ ID NO: 3), after anion-exchange chromatography of Example 1-1,without performing ultrafiltration/diafiltration.

In this regard, in order to link a maleimide reactive group at oneterminus of PEG of the mono-PEGylated immunoglobulin Fc region tocysteine of the GLP-1/GIP/Glucagon trigonal agonist analog 3, themono-PEGylated immunoglobulin Fc region was reacted with theGLP-1/GIP/Glucagon trigonal agonist analog 3 in a molar ratio of 1:1with a GLP-1/GIP/Glucagon trigonal agonist analog 3 concentration of 0.2g/L at 6° C.±4° C. for about 2 hours. The reaction was performed in aTris-Cl buffer (6° C.±4° C.) including isopropanol. As a result ofanalyzing the resultant after reaction using SE-HPLC, RP-HPLC, andIE-HPLC assays, the purity was confirmed to be 90% or more in SE-HPLC,80% or more in RP-HPLC, and 70% or more in IE-HPLC.

Thereafter, the resultant of the reaction was subjected to hydrophobicinteraction chromatography once using a Source 15ISO column (GEHealthcare). By-products were eliminated by this process, and animmunoglobulin Fc region-PEG-containing linker-GLP-1/GIP/Glucagontrigonal agonist analog 3 conjugate was obtained. In this case,purification was performed using a buffer including sodium citrate andan ammonium sulfate concentration gradient. It was confirmed that ayield obtained herein was increased by about twice or more compared to ayield of Comparative Example 1 with the same amount of theGLP-1/GIP/Glucagon trigonal agonist analog 3.

The eluted immunoglobulin Fc region-PEG-containinglinker-GLP-1/GIP/Glucagon trigonal agonist analog 3 conjugate wasanalyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity wasconfirmed since the purity was 90% or more in SE-HPLC, 90% or more inRP-HPLC, and 90% or more in IE-HPLC.

Example 2-4. Preparation of Conjugate of Glucagon Analog 1

A long-acting conjugate (immunoglobulin Fc region-PEG-containinglinker-Glucagon analog 1) was prepared via peptide conjugation ofGlucagon analog 1 (SEQ ID NO: 4), after anion-exchange chromatography ofExample 1-1, without performing ultrafiltration/diafiltration.

In this regard, in order to link a maleimide reactive group at oneterminus of PEG of the mono-PEGylated immunoglobulin Fc region toGlucagon analog 1, the mono-PEGylated immunoglobulin Fc region wasreacted with Glucagon analog 1 in a molar ratio of 1:1 with a Glucagonanalog 1 concentration of 0.2 g/L at 6° C.±4° C. for about 2 hours. Thereaction was performed in a Tris-Cl buffer (6° C.±4° C.) includingisopropanol. As a result of analyzing the resultant after reaction usingSE-HPLC, RP-HPLC, and IE-HPLC assays, the purity of the immunoglobulinFc region-PEG-containing linker-Glucagon analog 1 was confirmed to be90% or more in SE-HPLC, 70% or more in RP-HPLC, and 70% or more inIE-HPLC.

Thereafter, the resultant of the reaction was subjected to hydrophobicinteraction chromatography once using a Source 15ISO column (GEHealthcare). By-products were eliminated by this process, and animmunoglobulin Fc region-PEG-containing linker-Glucagon analog 1conjugate was obtained. In this case, purification was performed using abuffer including sodium citrate and an ammonium sulfate concentrationgradient. It was confirmed that a yield obtained herein was increased byabout 1.5 times or more compared to a yield of Comparative Example 2with the same amount of Glucagon analog 1.

The eluted immunoglobulin Fc region-PEG-containing linker-Glucagonanalog 1 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays,and high purity was confirmed since the purity was 90% or more inSE-HPLC, 90% or more in RP-HPLC, and 90% or more in IE-HPLC.

Example 2-5. Preparation of Conjugate of Glucagon Analog 2

A long-acting conjugate (immunoglobulin Fc region-PEG-containinglinker-Glucagon analog 2) was prepared via peptide conjugation ofGlucagon analog 2 (SEQ ID NO: 5), after anion-exchange chromatography ofExample 1-1, without performing ultrafiltration/diafiltration.

In this regard, in order to link a maleimide reactive group at oneterminus of PEG of the mono-PEGylated immunoglobulin Fc region toGlucagon analog 2, the mono-PEGylated immunoglobulin Fc region wasreacted with Glucagon analog 2 in a molar ratio of 1:1 with a Glucagonanalog 2 concentration of 0.2 g/L at 6° C.±4° C. for about 2 hours. Thereaction was performed in a Tris-Cl buffer (6° C.±4° C.) includingisopropanol. As a result of analyzing the resultant after reaction usingSE-HPLC, RP-HPLC, and IE-HPLC assays, the purity of the immunoglobulinFc region-PEG-containing linker-Glucagon analog 2 was confirmed to be90% or more in SE-HPLC, 70% or more in RP-HPLC, and 70% or more inIE-HPLC.

Thereafter, the resultant of the reaction was subjected to hydrophobicinteraction chromatography once using a Source 15ISO column (GEHealthcare). By-products were eliminated by this process, and animmunoglobulin Fc region-PEG-containing linker-Glucagon analog 2conjugate was obtained. In this case, purification was performed using abuffer including sodium citrate and an ammonium sulfate concentrationgradient. It was confirmed that a yield obtained herein was increased byabout 1.5 times or more compared to a yield of Comparative Example 2with the same amount of Glucagon analog 2.

The eluted immunoglobulin Fc region-PEG-containing linker-Glucagonanalog 2 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays,and high purity was confirmed since the purity was 90% or more inSE-HPLC, 90% or more in RP-HPLC, and 90% or more in IE-HPLC.

Example 2-6. Preparation of Conjugate of Glucagon Analog 3

A long-acting conjugate (immunoglobulin Fc region-PEG-containinglinker-Glucagon analog 3) was prepared via peptide conjugation ofGlucagon analog 3 (SEQ ID NO: 6), after anion-exchange chromatography ofExample 1-1, without performing ultrafiltration/diafiltration.

[SEQ ID NO: 6] YXQGTFTSDYSKYLDSRRAQDFVQWLMNTC

In this regard, in order to link a maleimide reactive group at oneterminus of PEG of the mono-PEGylated immunoglobulin Fc region tocysteine of Glucagon analog 3, the mono-PEGylated immunoglobulin Fcregion was reacted with Glucagon analog 3 in a molar ratio of 1:1 with aGlucagon analog 3 concentration of 0.2 g/L at 6° C.±4° C. for about 2hours. The reaction was performed in a Tris-Cl buffer (6° C.±4° C.)including isopropanol. As a result of analyzing the resultant afterreaction using SE-HPLC, RP-HPLC, and IE-HPLC assays, the purity of theimmunoglobulin Fc region-PEG-containing linker-Glucagon analog 3 wasconfirmed to be 90% or more in SE-HPLC, 70% or more in RP-HPLC, and 70%or more in IE-HPLC.

Thereafter, the resultant of the reaction was subjected to hydrophobicinteraction chromatography once using a Source 15ISO column (GEHealthcare). By-products were eliminated by this process, and animmunoglobulin Fc region-PEG-containing linker-Glucagon analog 3conjugate was obtained. In this case, purification was performed using abuffer including sodium citrate and an ammonium sulfate concentrationgradient. It was confirmed that a yield obtained herein was increased byabout 1.5 times or more compared to the existing yield with the sameamount of Glucagon analog 3.

The eluted immunoglobulin Fc region-PEG-containing linker-Glucagonanalog 3 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays,and high purity was confirmed since the purity was 90% or more inSE-HPLC, 90% or more in RP-HPLC, and 90% or more in IE-HPLC.

Tables 1 and 2 show comparison results between the methods thecomparative examples and examples.

TABLE 1 COMPARISON BETWEEN PREPARATION METHODS OF GLP-1/GIP/GlucagonTRIGONAL AGONIST ANALOG COMPARATIVE EXAMPLE EXAMPLE (CONVENTIONALPREPARATION METHOD) GLP-1/GIP/Glucagon GLP-1/GIP/GlucagonGLP-1/GIP/Glucagon GLP-1/GIP/Glucagon TRIGOAL AGONIST TRIGOAL AGONISTTRIGOAL AGONIST TRIGOAL AGONIST ANALOG 1 ANALOG 1 ANALOG 2 ANALOG 3REACTION REACTION 1] REACTION 1] Fc:PEG = 1:1 CONDITIONS Peptide:PEG =1:1.3 REACTION 2] PEG-Fc:Peptide = 1:1 REACTION 2]PEG-Peptide:IMMUNOGLOBULIN Fc REGION = 1:2 PURITY SE-HPLC 90% OR MORE90% OR MORE 90% OR MORE 90% OR MORE RP-HPLC 90% OR MORE 90% OR MORE 90%OR MORE 90% OR MORE IE-HPLC 90% OR MORE 90% OR MORE 80% OR MORE 90% ORMORE YIELD (WITH — TWICE OR MORE TWICE OR MORE TWICE OR MORE RESPECTHIGHER THAN HIGHER THAN HIGHER THAN TO PEPTIDE) COMPARATIVE COMPARATIVECOMPARATIVE EXAMPLE EXAMPLE EXAMPLE YIELD (WITH ABOUT 1.3 TIMES ABOUT1.3 TIMES ABOUT 1.3 TIMES RESPECT HIGHER THAN HIGHER THAN HIGHER THAN TOFc) COMPARATIVE COMPARATIVE COMPARATIVE EXAMPLE EXAMPLE EXAMPLE

TABLE 2 COMPARISON BETWEEN METHODS FOR PREPARING GLUCAGON ANALOGSCOMPARATIVE EXAMPLE (CONVENTIONAL PREPARATION METHOD) EXAMPLE (REVERSEORDER PREPARATION METHOD) GLUCAGON GLUCAGON GLUCAGON GLUCAGON ANALOG 1ANALOG 1 ANALOG 2 ANALOG 3 REACTION REACTION 1] REACTION 1] Fc:PEG = 1:1CONDITIONS Peptide:PEG = 1:1.3 REACTION 2] PEG-Fc:Peptid = 1:1 REACTION2] PEG-Peptide:IMMUNOGLOBULIN Fc REGION = 1:5 PURITY SE-HPLC 90% OR MORE90% OR MORE 90% OR MORE 90% OR MORE RP-HPLC 90% OR MORE 90% OR MORE 90%OR MORE 90% OR MORE IE-HPLC 90% OR MORE 90% OR MORE 90% OR MORE 90% ORMORE YIELD (WITH — 1.5 TIMES OR MORE 1.5 TIMES OR MORE 1.5 TIMES OR MORERESPECT HIGHER THAN HIGHER THAN HIGHER THAN TO PEPTIDE) COMPARATIVECOMPARATIVE COMPARATIVE EXAMPLE EXAMPLE EXAMPLE YIELD (WITH TIMES ORMORE TIMES OR MORE TIMES OR MORE RESPECT HIGHER THAN HIGHER THAN HIGHERTHAN TO Fc) COMPARATIVE COMPARATIVE COMPARATIVE EXAMPLE EXAMPLE EXAMPLE

The above description of the present invention is provided for thepurpose of illustration, and it would be understood by those skilled inthe art that various changes and modifications may be made withoutchanging technical conception and essential features of the presentinvention. Thus, it is clear that the above-described embodiments areillustrative in all aspects and do not limit the present invention.Therefore, the scope of the disclosure is defined not by the detaileddescription, but by the claims and their equivalents, and all variationswithin the scope of the claims and their equivalents are to be construedas being included in the disclosure.

1. A compound having a structure of Formula 2 below or a stereoisomer, asolvate, or a pharmaceutically acceptable salt thereof:

wherein in Formula 2 above, n is from 200 to
 250. 2. The compound or astereoisomer, a solvate, or a pharmaceutically acceptable salt thereofaccording to claim 1, wherein the immunoglobulin Fc region is derivedfrom IgG, IgA, IgD, IgE, or IgM.
 3. The compound or a stereoisomer, asolvate, or a pharmaceutically acceptable salt thereof according toclaim 2, wherein the immunoglobulin Fc region is derived from IgG1,IgG2, IgG3, or IgG4.
 4. The compound or a stereoisomer, a solvate, or apharmaceutically acceptable salt thereof according to claim 1, whereinthe immunoglobulin Fc region is in a dimeric form.
 5. The compound or astereoisomer, a solvate, or a pharmaceutically acceptable salt thereofaccording to claim 1, wherein the immunoglobulin Fc region comprises theamino acid sequence of SEQ ID NO:
 10. 6. The compound or a stereoisomer,a solvate, or a pharmaceutically acceptable salt thereof according toclaim 1, wherein the compound or a stereoisomer, a solvate, or apharmaceutically acceptable salt thereof is conjugated to aphysiologically active polypeptide, wherein the physiologically activepolypeptide is selected from the group consisting of glucagon-likepeptide-1 (GLP-1), granulocyte colony stimulating factor (G-CSF), humangrowth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growthhormone releasing hormone, growth hormone releasing peptide,interferons, interferon receptors, G-protein-coupled receptors,interleukins, interleukin receptors, enzymes, interleukin-bindingprotein, cytokine-binding protein, macrophage activating factor,macrophage peptide, B cell factor, T cell factor, protein A, allergyinhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumornecrosis factor, tumor suppressor, metastasis growth factor, α-1antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, highlyglycosylated erythropoietin, angiopoietins, hemoglobin, thrombin,thrombin receptor activating peptide, thrombomodulin, blood factors VII,VIIa, VIII, IX, and XIII, plasminogen activating factor, fibrin-bindingpeptide, urokinase, streptokinase, hirudin, protein C, C-reactiveprotein, lenin inhibitor, collagenase inhibitor, superoxide dismutase,leptin, platelet-derived growth factor, epithelial growth factor,epidermal growth factor, angiostatin, angiotensin, bone growth factor,bone stimulating protein, calcitonin, atriopeptin, cartilage inducingfactor, elcatonin, connective tissue activating factor, tissue factorpathway inhibitor, follicle stimulating hormone, luteinizing hormone,luteinizing hormone releasing hormone, nerve growth factor, parathyroidhormone, relaxin, secretin, somatomedin, insulin-like growth factor,adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrinreleasing peptide, corticotropin releasing factor, thyroid stimulatinghormone, autotaxin, lactoferrin, myostatin, incretins, gastricinhibitory polypeptide (GIP), GLP-1/GIP dual agonist, GLP-1/GIP/Glucagontrigonal agonist, cell surface antigens, virus derived vaccine antigens,monoclonal antibodies, polyclonal antibodies, and antibody fragments. 7.A composition for preparing a long-acting drug conjugate, comprising acompound having a structure of Formula 2 below, or a stereoisomer, asolvate, or a pharmaceutically acceptable salt thereof, wherein the drugis a physiologically active polypeptide:

wherein in Formula 2 above, n is from 200 to
 250. 8. The compositionaccording to claim 7, wherein the physiologically active polypeptide isselected from the group consisting of glucagon-like peptide-1 (GLP-1),granulocyte colony stimulating factor (G-CSF), human growth hormone(hGH), erythropoietin (EPO), glucagon, insulin, growth hormone releasinghormone, growth hormone releasing peptide, interferons, interferonreceptors, G-protein-coupled receptors, interleukins, interleukinreceptors, enzymes, interleukin-binding protein, cytokine-bindingprotein, macrophage activating factor, macrophage peptide, B cellfactor, T cell factor, protein A, allergy inhibitor, cell necrosisglycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumorsuppressor, metastasis growth factor, α-1 antitrypsin, albumin,α-lactalbumin, apolipoprotein-E, highly glycosylated erythropoietin,angiopoietins, hemoglobin, thrombin, thrombin receptor activatingpeptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII,plasminogen activating factor, fibrin-binding peptide, urokinase,streptokinase, hirudin, protein C, C-reactive protein, lenin inhibitor,collagenase inhibitor, superoxide dismutase, leptin, platelet-derivedgrowth factor, epithelial growth factor, epidermal growth factor,angiostatin, angiotensin, bone growth factor, bone stimulating protein,calcitonin, atriopeptin, cartilage inducing factor, elcatonin,connective tissue activating factor, tissue factor pathway inhibitor,follicle stimulating hormone, luteinizing hormone, luteinizing hormonereleasing hormone, nerve growth factor, parathyroid hormone, relaxin,secretin, somatomedin, insulin-like growth factor, adrenocorticalhormone, cholecystokinin, pancreatic polypeptide, gastrin releasingpeptide, corticotropin releasing factor, thyroid stimulating hormone,autotaxin, lactoferrin, myostatin, incretins, gastric inhibitorypolypeptide (GIP), GLP-1/GIP dual agonist, GLP-1/GIP/Glucagon trigonalagonist, cell surface antigens, virus derived vaccine antigens,monoclonal antibodies, polyclonal antibodies, and antibody fragments. 9.The composition according to claim 7, wherein the immunoglobulin Fcregion is derived from IgG1, IgG2, IgG3, or IgG4.
 10. The compositionaccording to claim 7, wherein the composition is used to prepare along-acting drug conjugate without performingultrafiltration/diafiltration in preparation of the long-acting drugconjugate.
 11. A method for preparing a long-acting conjugate of aphysiologically active polypeptide, the method comprising: preparing aconjugate by linking a compound having a structure of Formula 2 below ora stereoisomer, a solvate, or a pharmaceutically acceptable saltthereof, to a physiologically active polypeptide:

wherein in Formula 2 above, n is from 200 to
 250. 12. The methodaccording to claim 11, wherein the preparing of the conjugate isperformed by reacting the compound having a structure of Formula 2 belowor a stereoisomer, a solvate, or a pharmaceutically acceptable saltthereof with the physiologically active polypeptide in a molar ratio of1:1 to 1:3.
 13. The method according to claim 11, wherein the maleimideof the compound having a structure of Formula 2 below or a stereoisomer,a solvate, or a pharmaceutically acceptable salt thereof is linked to acysteine residue of the physiologically active polypeptide.
 14. Themethod according to claim 11, wherein the method is performed withoutultrafiltration/diafiltration after preparing the mono-PEGylatedimmunoglobulin Fc region.
 15. The method according to claim 11, furthercomprising purifying the conjugate by hydrophobic interactionchromatography.
 16. The method according to claim 11, wherein thephysiologically active polypeptide is selected from the group consistingof glucagon-like peptide-1 (GLP-1), granulocyte colony stimulatingfactor (G-CSF), human growth hormone (hGH), erythropoietin (EPO),glucagon, insulin, growth hormone releasing hormone, growth hormonereleasing peptide, interferons, interferon receptors, G-protein-coupledreceptors, interleukins, interleukin receptors, enzymes,interleukin-binding protein, cytokine-binding protein, macrophageactivating factor, macrophage peptide, B cell factor, T cell factor,protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin,lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growthfactor, α-1 antitrypsin, albumin, α-lactalbumin, apolipoprotein-E,highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin,thrombin receptor activating peptide, thrombomodulin, blood factors VII,VIIa, VIII, IX, and XIII, plasminogen activating factor, fibrin-bindingpeptide, urokinase, streptokinase, hirudin, protein C, C-reactiveprotein, lenin inhibitor, collagenase inhibitor, superoxide dismutase,leptin, platelet-derived growth factor, epithelial growth factor,epidermal growth factor, angiostatin, angiotensin, bone growth factor,bone stimulating protein, calcitonin, atriopeptin, cartilage inducingfactor, elcatonin, connective tissue activating factor, tissue factorpathway inhibitor, follicle stimulating hormone, luteinizing hormone,luteinizing hormone releasing hormone, nerve growth factor, parathyroidhormone, relaxin, secretin, somatomedin, insulin-like growth factor,adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrinreleasing peptide, corticotropin releasing factor, thyroid stimulatinghormone, autotaxin, lactoferrin, myostatin, incretins, GLP-1/GIP dualagonist, gastric inhibitory polypeptide (GIP), GLP-1/GIP/Glucagontrigonal agonist, cell surface antigens, virus derived vaccine antigens,monoclonal antibodies, polyclonal antibodies, and antibody fragments.17. The method according to claim 16, wherein the physiologically activepolypeptide is a GLP-1/GIP/Glucagon trigonal agonist, glucagon, or ananalog thereof.
 18. The method according to claim 17, wherein thephysiologically active polypeptide comprises any one amino acid sequenceof SEQ ID NOS: 1 to
 6. 19. The method according to claim 11, wherein theimmunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4. 20.A long-acting drug conjugate prepared using the method according toclaim
 16. 21. A long-acting drug conjugate prepared by reacting thecomposition according to claim 8 with a physiologically activepolypeptide.